ABSTRACT The emergence of antibiotic-resistant bacteria is one of the greatest threats to human health in the 21st century. In particular, vancomycin-resistant enterococci (VRE) are one of the most challenging organisms in clinical settings. Indeed, vancomycin-resistant Enterococcus faecium have been designated by the Infectious Disease Society of America as one of the ?superbugs? against which new therapies are urgently needed. Daptomycin (DAP), a cell membrane-targeting lipopeptide antibiotic with potent in vitro bactericidal activity against VRE, has become a key ?front-line? antimicrobial agent against these organisms. However, development of resistance during therapy is a daunting challenge. The major mediator of DAP resistance in enterococci is a cluster of genes (designated liaFSR) that encode a three-component regulatory system involved in orchestrating the cell envelope adaptive response to antibiotics and antimicrobial peptides. LiaR is the response regulator (LuxR-type) of the system whose activity seems to be regulated by LiaF and LiaS (histidine kinase). However, the specific regulatory role of LiaF in enterococci is unknown. Additionally, we have identified a cluster of three genes that are mediators of the LiaR response (designated liaXYZ). LiaX is a surface exposed and secreted protein that appears to be the main orchestrator of the LiaR- mediated cell membrane response by negatively regulating the LiaFSR system, controlling cell membrane phospholipid remodeling (a phenotype associated with DAP resistance). Additionally, the N-terminus of LiaX interacts with penicillin-binding protein 5 (a key enterococcal enzyme required for cell-wall synthesis in the presence of ?-lactams) and is likely to mediate the ?see-saw? effect (hypersusceptibility to ?-lactams upon developing of DAP resistance). LiaY and LiaZ are two transmembrane proteins that are regulated by LiaX. Our data indicate that LiaYZ are required for DAP resistance and that LiaY is likely responsible for changes in cell membrane phospholipid architecture. Thus, the overarching hypothesis of our proposal is that understanding the mechanisms by which LiaFSR and LiaXYZ orchestrate the cell membrane response against antibiotics would provide novel insights into the molecular mechanisms of antimicrobial peptide resistance and bacterial adaptation that could be exploited with novel therapeutic interventions. We plan to develop our experimental approach in three major specific aims. In Sp. Aim I, we will investigate of the role of LiaF, a transmembrane protein that seems to play a major and distinct role in the activation of the response regulator LiaR in enterococci. In Specific Aim II, we will focus on the characterization of LiaX as the master effector of the cell envelope adaptive response. Specific Aim III will assess the role LiaYZ as mediators of DAP resistance and cell membrane remodeling under the assumption that such effect is mediated through interactions with cardiolipin synthase, a major phospholipid enzyme. We expect to provide evidence for a novel biochemical paradigm to the cell envelope response to antibiotics and, potentially, new targets for drug development.